Transition metals such as Cu, Fe, Co have well known toxic effects. A large fraction of the transition metal uptake occurs through lysosomes and accumulation of transition metals in lysosomes was shown in the overload models. The effects of transition metals on lysosomal digestive machinery are not very well established. The preliminary data obtained for this project show that transition metals block ion channel mucolipin 1 (TRPML1). TRPML1 was previously identified as the lysosomal ion channel mutations in which cause lysosomal storage disease mucolipidosis type IV (MLIV). Similar to the effects of TRPML1 downregulation in MLIV, TRPML1 block by transition metals is associated with formation of cytoplasmic storage bodies, mitochondrial fragmentation and the loss of Ca2+ buffering by mitochondria. By analogy with the lysosome-mitochondria axis model of aging and cell death in lysosomal storage diseases, these data suggest that TRPML1 block by transition metals affects lysosomal function and, therefore autophagy. Suppressed autophagy results in accumulation of dysfunctional mitochondria that cannot take up cytoplasmic Ca2+ and guard cells against pro-apoptotic effects of Ca2+ spikes. The central premise of this proposal is: if TRPML1 block contributes to transition metals toxicity, then all transition metals that block TRPML1 should induce the same MLIV-like phenotype. This novel model of the lysosomal contribution to transition metal toxicity will be tested using several approaches. First, the range of transition metals that block TRPML1 will be established in order to correlate TRPML1 block by metals and lysosomal storage phenotype induced by such block. Whether or not all transition metals that block TRPML1 induce autophagy suppression and accumulation of mitochondria that are compromised in their Ca2+ buffering function will be established. Structural determinants of TRPML1 block by transition metals will be established and transition metal resistant mutants will be created. Such mutants will be used to replace native TRPML1 and establish the relative contribution of TRPML1 block into lysosomal toxicity of transition metals. The results obtained in the course of this project are expected to identify a novel link between lysosomal buildup of transition metals and cell death and may provide a rationale for novel pharmacological approaches to transition metals toxicity. The basic biological implications of this project include better understanding of the effect of toxic metals, structure and function of ion channels and housekeeping role of lysosomes. PUBLIC HEALTH RELEVANCE: The present project aims to answer whether the block of the lysosomal ion channel TRPML1 contributes to transition metal toxicity. Delineating the chain of events that connects lysosomal accumulation of transition metals to cell death will pave way for a detailed inquiry into lysosomal aspects of transition metal toxicity and may suggest novel pharmacological approaches to complement the existing treatments for metal toxicity. Since lysosomes are involved in the organism-level functions such as antigen processing, proving that transition metals affect lysosomal function may have even broader biological impact.